Dot1L interacts with Zc3h10 to activate UCP1 and other thermogenic genes
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Abstract
Brown adipose tissue is a metabolically beneficial organ capable of dissipating chemical energy into heat, thereby increasing energy expenditure. Here, we identify Dot1L, the only known H3K79 methyltransferase, as an interacting partner of Zc3h10 that transcriptionally activates the UCP1 promoter and other BAT genes. Through a direct interaction, Dot1L is recruited by Zc3h10 to the promoter regions of thermogenic genes to function as a coactivator by methylating H3K79. We also show that Dot1L is induced during brown fat cell differentiation and by cold exposure and that Dot1L and its H3K79 methyltransferase activity is required for thermogenic gene program. Furthermore, we demonstrate that Dot1L ablation in mice using UCP1-Cre prevents activation of UCP1 and other target genes to reduce thermogenic capacity and energy expenditure, promoting adiposity. Hence, Dot1L plays a critical role in the thermogenic program and may present as a future target for obesity therapeutics.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 11 2020, follows.
Summary
In this article, the authors sought to identify interacting partner(s) of Zc3h10, a transcription factor that activates expression of UCP-1 and other brown-adipocyte-specific genes. They identified the H3K79 methyltransferase Dot1L as an epigenetic modifier that interacts with Zc3h10 and facilitates its action on UCP-1 and other brown-adipocyte-specific genes. The strength of the manuscript is that the hypotheses were examined by a wide range of approaches, including protein-protein interaction assays, cell culture studies, and animal models. The present data provide solid evidence for an important role of Dot1L in the regulation of brown-adipocyte-specific genes. At the same time, all of the reviewers …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 11 2020, follows.
Summary
In this article, the authors sought to identify interacting partner(s) of Zc3h10, a transcription factor that activates expression of UCP-1 and other brown-adipocyte-specific genes. They identified the H3K79 methyltransferase Dot1L as an epigenetic modifier that interacts with Zc3h10 and facilitates its action on UCP-1 and other brown-adipocyte-specific genes. The strength of the manuscript is that the hypotheses were examined by a wide range of approaches, including protein-protein interaction assays, cell culture studies, and animal models. The present data provide solid evidence for an important role of Dot1L in the regulation of brown-adipocyte-specific genes. At the same time, all of the reviewers believed that the mechanisms involved in the epigenetic regulation of gene expression by Dot1L need to be investigated in more depth. Because the roles and the regulation of H3K79 methylation are still not fully understood, it would advance the field if the authors could provide additional data along these lines.
Essential Revisions
The protein expression of Dot1L appears disproportionally specific to brown adipose tissue compared with the mRNA expression. Is there a protein-gene difference? Is the identity of the band correct? Can the author see the loss of the band in the BKO tissues?
Most of the experiments appear to have been performed under basal conditions (e.g. unstimulated cells, room temperature mice). Dot1L expression increases with cold exposure and in their previous study that Zc3h10 is recruited to thermogenic gene promoters by p38 MAPK phosphorylation in response to adrenergic activation. The present study would benefit from additional experiments demonstrating a dynamic role for Dot1L in thermogenic gene transcription during cold exposure. Does cold exposure/adrenergic activation modulate Dot1L-Zc3h10 interaction, Dot1L recruitment to thermogenic gene promoters, H3K79 methylation, etc. in a p38 MAPK-dependent manner?
The authors conclude that "these results show that Dot1L methyltransferase activity is required for thermogenic gene expression and other Zc3h10 target genes for the BAT gene program (line 175)" and that "Dot1L enzymatic activity is critical for activating the thermogenic gene program in vivo" (line 255). These statements are not yet strongly supported. The involvement of H3K79 methylation was implied through the use of a chemical inhibitor EPZ5676 (figure 2c and figure 3) and the reduction of H3K79me3 levels in whole-cell lysates (figure 3b). Can the authors confirm the change of H3K79 methylation at brown-adipocyte-specific genes by conducting ChIP-QPCR in the experiments where they modulate either expression or enzymatic activity of Dot1L? In addition the authors could compare the effect of the overexpression of wild-type Dot1L (figure 2d) with a mutant Dot1L in which critical residue(s) are substituted in the enzyme catalytic core.
In Figure 4D, ChIP-qPCR shows the reduction of H3K79me2 and H3K79me3 enrichment on thermogenic gene promoters in Dot1L knockout BAT. It would be informative to examine changes in Dot1L and Zc3h10 binding on the same regions in BAT.
Although not absolutely required, it would be ideal to look at global epigenetic changes following induction or inhibition of Zc3h10 and/or Dot1L. Of particular interest is whether Zc3h10 plays any role in tethering Dot1L and modulating H3K79 methylation at brown-adipocyte-specific genes and whether the epigenetic changes induced by are specific to brown adipocyte function or not. The authors should consider performing ChIP-seq for H3K79me2/3 and Zc3h10. The use of tagged protein is an alternative if the antibody is not available for the latter. H3K79me2/3 reportedly marks a subset of enhancers (2019 Nat Commun, 10.1038/s41467-019-10844-3). It is also of interest whether such H3K79me2/3-marked enhancers are enriched in the vicinity of the brown-adipocyte-specific genes.
The raw ATAC-seq data show very high background (figure 5A). How many peak calls were obtained? Improvement is necessary to discuss the qualitative differences. Please consider the use of culture cells if the technical hurdle is caused by the use of tissue samples. Further, the pattern of the aggregate plots (figure 5A left upper) does not obviously match that of the heatmaps (figure 5A, left lower). Please describe exactly what is shown in the figure. Are the scales the same for each panel or the heatmaps zoom in the area indicated by the red dotted boxes in the aggregate plot? What do the red dotted boxes mean? What does color scale bar in the middle heatmap mean?
Also related the ATAC data, Figure 5C suggests that loss of Dot1L alters chromatin accessibility at far more genes than is represented in the "Shared processes" pathway analysis. It would be helpful to see a more comprehensive analysis of the ATAC-seq data (is thermogenesis one of the top pathways, what other pathways are affected, is the Zc3h10 binding motif overrepresented at sites of increased chromatin accessibility? etc.) and discussion about why the ATAC-seq changes might be more general/less specific than the RNA-seq changes.
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